Rejection of organ allografts is dependent on T cells. It is now known that T cell stimulation through the T cell receptor (TCR), while required for antigen-specific activation, is not by itself sufficient, as a second (or "costimulatory") signal is also needed. In fact, TCR stimulation in the absence of a second signal can induce a state of anergy in the T cell, rendering it refractory to subsequent stimulation. The best-characterized costimulatory pathway occurs through interaction of CD28 found on the surface of T cells with either of two ligands, B7-1 or B7-2, found on activated antigen presenting cells. A soluble fusion protein called CTLA4Ig has been developed which is capable of binding to B7-1 and B7-2, blocking the delivery of costimulatory signals to T cells through CD28. Using a cardiac allograft model, we have shown that the combination of donor-specific transfusion (DST) plus CTLA4Ig, blocks rejection and induces long-term graft survival. Interestingly, CTLA4Ig is most effective when its administration is delayed for 2 days post-transplant. When given on the day of transplantation, CTLA4Ig only rarely induces long-term engraftment. Thus delaying T cell costimulatory blockade until after antigenic challenge is essential for tolerance induction. This application will examine the mechanism(s) by which DST plus CTLA4Ig act and the utility of this strategy. In aim #1, we will determine if DST serves merely as a source of antigen, or if DST-induces chimerism which is required for tolerance. These studies will use extracted soluble histocompatibility antigens as well as synthetic MHC-derived peptides as alternative sources of antigen. The ability of DST to induce chimerism, and the identity of chimeric cells, will be assessed by flow cytometry and PCR. The need for chimerism will be determined by using DST cells containing the herpesvirus thymidine kinase gene, and subsequently deleting these cells in vivo with ganciclovir. In aim#2 we will investigate the mechanisms of tolerance induction (deletion, anergy, suppression). Deletion will be assessed with in vivo immune responses to antigen where responding T cells can be identified by specific anti- TCRVbeta and V-alpha mAbs. In these models, we will track the fate of antigen-reactive cells in animals treated with CTLA4Ig. If responding T cells are deleted, we will study if CTLA4Ig induces apoptosis by preventing expression of cell survival genes such as bcl-2 and/or bcl-x. To test for anergy we attempt to reverse unresponsiveness with exogenous cytokines or infection. Suppression will be investigated with adoptive transfer studies. Lastly, as there are theoretical reasons why the effects of the CTLA4Ig + DST regimen might be antagonistic with those of cyclosporine, in aim #3 we will determine how CTLA4Ig interacts with standard immunosuppressive agents such as cyclosporine and prednisone in an in vivo transplant model. Through these studies, we hope to learn more about the use of DST in tolerance induction, the mechanism of action of DST and CTLA4Ig, and how best to use CTLA4Ig clinically. These studies also should help lead to the design of new immunosuppressive strategies in the future.